A prothrombin activator from Bothrops erythromelas (jararaca-da-seca) snake venom: characterization and molecular cloning

Abstract

A novel prothrombin activator enzyme, which we have named 'berythractivase', was isolated from Bothrops erythromelas (jararaca-da-seca) snake venom. Berythractivase was purified by a single cation-exchange-chromatography step on a Resource S (Amersham Biosciences) column. The overall purification (31-fold) indicates that berythractivase comprises about 5% of the crude venom. It is a single-chain protein with a molecular mass of 78 kDa. SDS/PAGE of prothrombin after activation by berythractivase showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin, independent of the prothrombinase complex. Chelating agents, such as EDTA and o -phenanthroline, rapidly inhibited the enzymic activity of berythractivase, like a typical metalloproteinase. Human fibrinogen A alpha-chain was slowly digested only after longer incubation with berythractivase, and no effect on the beta- or gamma-chains was observed. Berythractivase was also capable of triggering endothelial proinflammatory and procoagulant cell responses. von Willebrand factor was released, and the surface expression of both intracellular adhesion molecule-1 and E-selectin was up-regulated by berythractivase in cultured human umbilical-vein endothelial cells. The complete berythractivase cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 2330 bp and encodes a preproprotein with significant sequence similarity to many other mature metalloproteinases reported from snake venoms. Berythractivase contains metalloproteinase, desintegrin-like and cysteine-rich domains. However, berythractivase did not elicit any haemorrhagic response. These results show that, although the primary structure of berythractivase is related to that of snake-venom haemorrhagic metalloproteinases and functionally similar to group A prothrombin activators, it is a prothrombin activator devoid of haemorrhagic activity. This is a feature not observed for most of the snake venom metalloproteinases, including the group A prothrombin activators.

An overview of Bothrops erythromelas venom

“…Schattner et al (22) complemented the results reported by Silva et al (18) by evaluating the effects of two P-III snake venom metalloproteinases (SVMPs), berythrativase and jararhagin, isolated from B. erythromelas and B. jararaca, respectively, on HUVECs. Both SVMPs were capable of stimulating the releases of interleukin-8 (IL-8), nitric oxide, and prostacyclin (PGI 2 ) by the HUVECs.…”

“…In 2006, Pereira et al (31) studied berythrativase and jararhagin to complement the studies conducted by Schattner et al (22) and Silva et al (18) . Pereira et al (31) studied the differences in the biological effects of the two SVMPs on different hemostatic properties.…”

“…Silva et al (18) were the fi rst to conduct a study involving the molecular cloning, purifi cation, and characterization of a prothrombin activator of B. erythromelas. Their fi ndings complemented those obtained by Nahas et al (8) , and they successfully purifi ed the prothrombin activator berythrativase, by single cation-exchange chromatography.…”

rACLF, a recombinant snake venom metalloprotease, activates endothelial cells in vitro

“…SVMPs are divided into four groups (PI, II, III and IV) and subgroups that differ for the presence of additional domains on the carboxyl side of the metalloproteinase domain (4,12,17). SVMPs display several activities such as degradation of blood coagulating factors (7,30), extracellular matrix (ECM) components (4,14,23,26) and cell receptors such as the cell-surface β1-integrin receptor (18). Besides these activities, SVMPs were reported to induce apoptosis in endothelial cells (2,32).…”

Ação da jararagina na expressão gênica e protéica de mediadores pró-inflamatórios por células endoteliais humanas.

“…Nessa reclassificação, a classe P-IV foi incuída na classe P-III por ser considerada como o resultado de uma modificação pós-traducional da classe P-III. SILVA et al, 2003;WANG et al, 2008), proteínas de membrana celular e à interação com receptores específicos, como integrinas de superfície de plaquetas (DE LUCA et al, 1995;YOU et al, 2006), células endoteliais (SOUZA et al, 2000;COMINNETTI et al, 2004) e fibroblastos (ZIGRINO et al, 2002), ativando ou inibindo a resposta celular. Esses efeitos podem resultar em várias alterações fisiopatológicas tais como inflamação, inibição da agregação plaquetária, apoptose e hemorragia .…”